Project 4 ? Project Summary The innate immune response helps maintain a balanced homeostatic relationship with the commensal gut microbiota, but importantly has potent indirect effects on host physiology. Gut microbiota immunomodulatory actions impact bone remodeling, but the mechanisms are unclear. Bone remodeling is the continuous process in which the skeleton is dynamically renewed by the actions of bone resorbing osteoclasts and bone forming osteoblasts. Skeletal deterioration occurs when the action of the osteoclasts exceeds those of the osteoblasts. Young-adult germfree (GF) mice have increased bone mass, which is reversed upon colonization with gut microbiota from specific pathogen free (SPF) mice. Commensal microbiota in SPF mice enhance osteoclastogenesis and suppress osteoblastogenesis, which drives bone loss. Intriguingly, gut microbiota effects on bone remodeling do not appear to be mediated by immune responses within the gut, but rather reflect communication between the gut and liver. When compared to GF mice, SPF mice have increased inflammatory cytokines in the liver, but not in the ileum. SPF mice also have enhanced inflammatory monocytes in liver draining lymph nodes, but not the mesenteric lymph nodes. These findings support a novel signaling axis between the gut and liver that regulates bone remodeling. The scientific premise is supported by preliminary data showing that Myd88-dependent signaling components were increased and hepatocyte-associated innate immune factors were upregulated in the livers of SPF compared to GF mice. Commensal gut microbiota increased liver and serum levels of lipocalin-2 (LCN2), which is a candidate innate immune factor that controls bone remodeling. This research will test the overall hypothesis that circulating gut microbiota-derived ligands stimulate liver cell MyD88-dependent synthesis of innate immune serum factors that regulate bone remodeling. Hepatocyte-specific null and myeloid-specific null mice will be housed under GF and SPF conditions, and proteomics, microbiome sequencing, and osteoimmunology research techniques will be applied. Aim 1 will test the hypothesis that the gut microbiota stimulates liver innate immunity and the synthesis of related serum factors through MyD88- dependent signaling in hepatocytes and liver macrophages. Aim 2 will test the hypothesis that gut microbiota stimulation of hepatocyte-derived LCN2 enhances osteoclastogenesis and suppresses osteoblastogenesis.!The proposed studies will determine the relationship between the commensal gut microbiota, liver innate immune response, and bone remodeling. This research is clinically relevant because the mechanisms driving continuous bone loss in the young adult skeleton are unknown. Individuals with gut and liver disorders have an increased prevalence of osteoporosis, which further underscores the relationship between the gut, liver and bone.